Note Algae 2021, 36(2): 1-11 https://doi.org/10.4490/algae.2021.36.5.6 Open Access First record of the cyanobacterial genus Wilmottia (Coleofascicula- ceae, Oscillatoriales) from the South Orkney Islands (Antarctica) Ranina Radzi1, Faradina Merican1,2,*, Paul Broady3, Peter Convey4, Narongrit Muangmai5,6 Wan Maznah Wan Omar1,2 and Sébastien Lavoué1 1School of Biological Sciences, Universiti Sains Malaysia, Minden, 11800 Penang, Malaysia 2National Antarctic Research Centre, University of Malaya, 50603 Kuala Lumpur, Malaysia 3School of Biological Sciences, University of Canterbury, Christchurch 8041, New Zealand 4British Antarctic Survey, NERC, Cambridge CB3 0ET, UK 5Faculty of Fisheries, Kasetsart University, Bangkok 10900, Thailand 6Graduate School of Integrated Science for Life, Hiroshima University, Hiroshima 739-0046, Japan Two cyanobacterial morphotypes isolated from Signy Island, South Orkney Islands, maritime Antarctica were char- acterised using a polyphasic approach combining morphological, cytological and molecular analyses. These analyses showed that the strains grouped with members of the genus Wilmottia. This genus has three species, W. murrayi, W. stricta, and W. koreana. Both morphotypes analysed in this study were placed within the clade of W. murrayi. This clade showed a well-supported separation from Antarctica and New Zealand strains, as well as the strains from other regions. W. murrayi was first described from Antarctica and is now known from several Antarctic regions. Confirmation of the occurrence of W. murrayi at Signy Island significantly extends its known distribution in Antarctica. In addition, a new combination, W. arthurensis, is suggested for Phormidium arthurensis. Key Words: Antarctic cyanobacteria; distribution; polyphasic analysis; taxonomy; 16S rDNA INTRODUCTION The genus Wilmottia O. Strunecky, J. Elster & J. Ko- 2004, Casamatta et al. 2005, Taton et al. 2006, Comte et al. márek was established from material originally referred 2007) to various soil conditions (Šabacká and Elster 2004, to Phormidium Kützing ex Gomont that originated from Komárek and Anagnostidis 2005, Comte et al. 2007), stag- Antarctica. According to Strunecký et al. (2011), the ge- nant to slightly saline water (Komárek and Anagnostidis nus Wilmottia originally comprised a single species, W. 2005), seepages (Šabacká and Elster 2004, Comte et al. murrayi (West & G. S. West) Strunecký, Elster & Komárek, 2007), and even on whale bones (Strunecký et al. 2010). which was described based on a polyphasic evaluation The species has also been recorded from New Zealand of Phormidium-like strains isolated from Antarctic lo- rivers (Wood et al. 2009, Heath et al. 2010) and on wet calities. Subsequently, W. murrayi has been recorded rocks on the seashore in Brazil (Lokmer 2007). A second from diverse habitats around the Antarctic, ranging from species, W. stricta Machado-de-Lima, Martins et Branco freshwater lakes (Boyer et al. 2002, Šabacká and Elster has been described from streams of São Paulo, Brazil This is an Open Access article distributed under the Received November 30, 2020, Accepted May 6, 2021 terms of the Creative Commons Attribution Non-Com- Corresponding Author mercial License (http://creativecommons.org/licenses/by-nc/3.0/) which * permits unrestricted non-commercial use, distribution, and reproduction E-mail: [email protected] in any medium, provided the original work is properly cited. Tel: +604-653-5878, Fax: +604-656-5125 Copyright © 2021 The Korean Society of Phycology 1 http://e-algae.org pISSN: 1226-2617 eISSN: 2093-0860 Algae 2021, 36(2): 1-11 (Machado-de-Lima et al. 2017). The genus Wilmottia was initially included within the A family Phormidiaceae, but it was later transferred to Co- leofasciculaceae based on molecular and ultrastructural evidence (Komárek et al. 2014). Species are described as having solitary trichomes or with several to many trichomes forming fascicles, presenting cylindrical tri- chome with cells almost isodiametric or longer than wide that are not or only slightly constricted at cross-walls (Komárek and Anagnostidis 2005). Solitary granules are present in vegetative cells and are occasionally placed near the cross-walls (Komárek and Anagnostidis 2005). The apical cell is rounded or slightly conical without any distinctive feature, which conforms to Phormidium group IV (Komárek and Anagnostidis 2005). The genus B as currently described possesses characteristics that are morphologically identical to those of Phormidium, thus hindering reliable morphological assessment. Hence, representatives of this genera can only be distinguished with confidence based on the molecular evaluation. In a broad-scale, floristic survey conducted on Signy Island (South Orkney Islands, 60.7170° S, 45.6000° W) by Broady (1979) recorded the occurrence of fivePhormidi - um morphospecies: P. autumnale Gomont, P. foveolarum Gomont, P. frigidum F. E. Fritsch, P. priestleyi F. E. Fritsch, Fig. 1. Map showing the sample locations on Signy Island, South and P. cf. rubroterricola N. L. Gardner. These identifica- Orkney Islands, Antarctica. (A) Location of Signy Island, South Orkney tions were based solely on morphological features and Islands, in relation to the Antarctic Peninsula and southern South Signy Island isolates have not been subjected to any America. (B) Enlargement which more accurately locates sample loca- tions that are also shown within the box on the main figure. Samples subsequent molecular assessment. However, recent mo- A and B were obtained at the northern end of the lateral moraine lecular evaluation of P. priestleyi resulted in the proposi- of Orwell Glacier in Cemetery Bay and on the slope north-west of tion of the new genus Phormidesmis Turicchia, Ventura, Waterpipe Beach, respectively. Map prepared by Laura Gerrish (BAS Komárková & Komárek with Phormidesmis priestleyi (F. E. Mapping and Geographic Information Centre). Fritsch) Komárek, Kastovský, Ventura, Turicchia & Smar- da as the type species (Komárek et al. 2009). This study il- lustrated the potential of genetic data to complement the B (60°42.020′ S, 45°36.480′ W) on the slope north-west of morphological approach in determining which species Waterpipe Beach (Fig. 1). Both samples were collected belong to the genus Phormidium. In the current study, from rock surfaces using sterile equipment. Sample A we report the polyphasic characterization of two cyano- comprised of moss while sample B comprised of lichens bacterial morphotypes that resemble P. cf. rubroterricola and moss. They were frozen soon after collection at the as described by Broady (1979). research station of the British Antarctic Survey on Signy Island and transported frozen (-20°C) in sterile contain- ers to Universiti Sains Malaysia, Penang, Malaysia. A MATERIALS AND METHODS small amount of each sample was streak inoculated onto 1% agarised full-strength BG-11 medium in Petri plates Sample origin and culture conditions (Rippka et al. 1979), supplemented with 1% cyclohexi- mide to prevent undesirable growth of eukaryotes (Bolch Two samples were obtained from Signy Island during and Blackburn 1996). Cultures were incubated for three the austral summer of 2015/16: (1) sample A (60°42.570′ weeks under 24 h light provided by a cool white fluores- S, 45°36.550′ W) at the northern end of the lateral mo- cent lamp at 27 µmol m-2 s-1 at 15 ± 2°C. Unialgal cultures raine of Orwell Glacier in Cemetery Bay; and (2) sample of filamentous cyanobacteria were established by inocu- https://doi.org/10.4490/algae.2021.36.5.6 2 Radzi et al. Cyanobacterial Genus Wilmottia lation onto fresh medium of small amounts of filaments µg mL-1 between 12 and 24 h. The 16S rDNA gene se- removed from the edges of developing colonies. quences were amplified using the combination of prim- ers 2 (5′-GGG GGA TTT TCC GCA ATG GG-3′) and 3 (5′- Morphological characterization CGC TCT ACC AAC TGA GCT A-3′) (Boyer et al. 2001). The reaction mix comprised 25 µL of MyTaq™ Red Mix Morphological examination was carried out on the (Meridian Bioscience, Cincinnati, OH, USA), 1 µL of each strains in cultures using an Olympus BX-53 bright field forward and reverse primer, 2 µL of DNA template, and microscope (Olympus America Inc., Center Valley, PA, 21 µL of ultra-pure water, giving a final volume of 50 µL. USA) at 100–2,000× magnification. Photomicrographs Polymerase chain reaction (PCR) was carried out using were taken. A camera lucida was used for making the il- a Thermal Cycler (Bio-Rad, Hercules, CA, USA). Thermal lustrations. The following morphological features were cycling conditions were set as follows: 95°C for 2 min, recorded for both morphotypes: filament and trichome 95°C for 15 s, 55°C for 15 s (30 cycles), 72°C for 20 s fol- width, cell shape, apical cell shape, sheath morphology, lowed by 7 min extension at 72°C. Once the reaction was cell colour, and presence of granules. Measurements completed, the integrity of the PCR product was verified were taken from 30 replicates of randomly chosen for using 2% agarose gel. PCR products were sequenced at every specimens. The characteristics of the studied mor- NHK Bioscience Solutions Sdn. Bhd. (Korea) using the photypes were compared with data presented in Broady Sanger sequencing method and the same primer pair (1979), Komárek and Anagnostidis (2005), Strunecký et used for PCR reactions. al. (2011), and Machado-de-Lima et al. (2017). Phylogenetic analyses Transmission electron microscopy The chromatograms were checked, edited, and Filaments taken from week three cultures of both mor- trimmed manually to achieve maximum homology. photypes were fixed for 24 h in McDowell-Trump fixative The 16S rDNA consensus sequences were built in by as- solution (McDowell and Trump 1976) prepared in 0.1 M sembling the forward and reverse sequences. Sequence phosphate buffer, and then post-fixed with %1 osmium alignments between newly determined sequences and a tetroxide. Upon fixation, the samples were dehydrated selection of GenBank sequences, were made using MUS- in an ethanol series (50, 75, 95, and 100%), 50 and 75% CLE algorithm in the Geneious 11.0 software package ethanol for 15 min each, followed by 2 times dehydration (http://www.geneious.com) (Kearse et al.